UNIVERSITY    OF   CALIFORNIA    PUBLICATIONS 

IN 

AGRICULTURAL   SCIENCES 

Vol.  2,  No.  8,  pp.  243-248,  plate  44  September  17,  1924 


MICROSPOROGENESIS  OF  GINKGO  BILOBA  L. 

WITH  ESPECIAL  REFERENCE  TO 

THE  DISTRIBUTION  OF  THE 

PLASTIDS   AND   TO   CELL 

WALL  FORMATION 

BY 

MARGARET  CAMPBELL  MANN 

(Contribution  from  the  Division  of  Genetics,  University  of  California) 


Microsporogenesis  in  Ginkgo  biloba  L.  is  especially  interesting 
because  the  plastids  are  definitely  oriented  with  respect  to  the  division 
figures  and  because  they  are  distributed  so  that  each  pollen  cell  receives 
approximately  one-fourth  of  them.  The  cells  are  large,  and  both 
plastids  and  chromosomes  can  be  observed  in  living  cells.  The  12 
pairs  of  chromosomes  are  nicely  separated  at  late  prophase,  and  one 
of  them  is  twice  as  large  as  the  others.  This  point  was  previously  noted 
by  Cardiff  (1906)  and  Ishikawa  (1910). 

Smears  stained  in  aceto-carmine  were  used  for  most  of  this  study, 
but  smears  were  also  fixed  in  Plemming  's  weak  and  chrom-acetic-urea 
and  stained  in  iron-haematoxylin,  Flemming's  triple,  and  safranin  and 
light  green.  The  plastids  are  easily  observed  in  aceto-carmine  since 
the  large  starch  grains  resist  the  carmine,  remaining  a  transparent 
green  while  the  rest  of  the  cytoplasm  stains  pink,  and  the  chromosomes 
bright  red.  The  starch  grains  stain  a  deep  blue  in  iodine,  but  show  no 
color  in  Flemming  's  triple  or  haematoxylin.  With  the  former  a  layer 
of  gentian,  with  the  latter  a  layer  of  bluish-black  cytoplasm,  surrounds 
each  starch  grain.  Unless  one  had  examined  the  pollen  mother  cells 
before  fixation,  he  might  easily  interpret  the  plastid-bearing  area  in 
fixed  material  as  an  unusually  coarse  cytoplasmic  mesh.  This  is 
probably  the  reason  why  the  phenomena  described  below  have  not  been 
previously  observed. 


244  University  of  California  Publications  in  Agricultural  Sciences       [Vol.  2 

The  two  nuclear  divisions  precede  cell  division  as  in  many  of  the 
higher  plants.  It  will  be  seen  from  the  account  which  follows  that  the 
reduction  division  is  typical  in  all  respects. 

Before  the  first,  or  so-called  heterotypic  division  occurs,  the  plastids 
lie  between  the  wall  and  the  nucleus  (figs.  1  and  2).  As  the  prophase 
progresses  they  enlarge,  and  some  of  them  divide.  The  division  is 
usually  a  simple  bipartition,  but  some  appearances  suggestive  of  bud- 
ding were  seen.  The  cell  wall  is  very  thin  at  first  but  gradually 
thickens  during  the  first  division.  The  plastids  remain  between  the 
nucleus  and  the  wall  until  after  the  first  spindle  has  disappeared,  when 
they  gradually  move,  or  more  probably  are  moved,  between  the  two 
groups  of  late  anaphase  chromosomes,  there  forming  a  ring  which 
nearly  fills  the  space  between  the  two  nuclei  (fig.  4).  This  position 
is  retained  until  after  the  second  division.  In  the  late  second  ana-  or 
early  telophase  a  portion  of  the  ring  of  plastids  is  drawn  between  each 
of  the  two  sets  of  daughter  nuclei  (fig.  7).  The  ring  then  breaks  in 
four  places,  so  that  one  quarter  of  it,  and  consequently  about  a  fourth 
of  the  plastids,  come  to  surround  the  inner  face  of  each  nucleus  (figs. 
7  and  8).  The  outer  wall  now  pushes  inward  between  each  of  the  four 
nuclei  and  finally  separates  the  pollen  cells  (fig.  9) .  Before  the  inpush- 
ing  of  the  outer  wall  two  cell  plates  form  at  right  angles  to  each  other. 
The  method  of  cell  division  in  Ginkgo,  then,  is  a  combination  of  cell 
plate  formation  and  cytokinesis.  The  inner  portion  of  the  wall  (which 
lies  next  to  the  plastid-bearing  area)  thickens  greatly  while  the  outer 
wall  remains  unchanged.  As  the  inner  wall  attains  its  final  thickness, 
the  plastids  become  generally  distributed  between  it  and  the  nucleus, 
and  a  new  wall,  staining  in  gentian-violet,  appears  about  each  pollen 
cell.  The  old  wall  stains  in  orange  or  in  light  green.  As  the  pollen 
cells  grow  they  burst  the  thin  outer  walls  of  this  case,  leaving  an  empty 
shell.  The  plastids  now  appear  smaller,  since  the  amount  of  starch  in 
them  is  considerably  reduced. 

Juranyi  (1872,  pi.  31,  fig.  13)  figured  a  light  band  between  the  two 
daughter  nuclei  of  the  pollen  mother  cells  of  Ceratozamia,  which  is 
very  like  the  position  of  the  starch  grains  of  Ginkgo  at  this  stage. 
Sprecher  (1907,  p.  155)  figured  the  cell  plate  formation  of  the  pollen 
mother  cells  of  Ginkgo,  but  neither  figures  nor  mentions  plastid  or 
starch  grain  distribution.  Moore  (1903)  figured  plastids  like  those  of 
Ginkgo  in  the  pollen  mother  cells  of  Pallavincinia.  but  does  not  discuss 
their  distribution.     Smith   (1907)   shows  starch  grains  in  the.  pollen 


1924]  Mann:  Microsporogenesis  of  Ginkgo  biloba  L.  245 

tube  of  Cycas  which  greatly  resemble  those  seen  in  the  pollen  mother 
cells  of  Ginkgo. 

The  procedure  described  above  raises  a  number  of  interesting 
questions.  Firstly,  the  position  of  the  starch  grains  bears  a  definite 
relation  to  the  formation  of  the  cell  walls.  They  are  generally  dis- 
tributed while  the  first  wall  is  forming,  and  it  ceases  to  thicken  when 
they  withdraw.  They  lie  near  the  forming  cell  plate,  and  near  the 
thick  inner  wall  during  its  formation.  Finally,  they  become  generally 
distributed  during  the  formation  of  the  pollen  cell  wall.  They  are  also 
smaller  than  they  were  during  the  formation  of  the  thick  inner  wall 
of  the  pollen  case.  It  seems  possible  that  they  may  provide  the  reserve 
material  which  is  utilized  in  wall  formation. 

Secondly,  the  method  of  cell  wall  formation  differs  from  that 
common  to  the  higher  plants.  This  is  of  particular  interest  on  account 
of  the  phylogenetic  position  of  Ginkgo. 

The  changes  of  position  of  the  starch  grains  are  essentially  the  same 
as  those  noted  by  Terni  (1914)  for  the  chondriosomes  in  the  spermato- 
genesis of  Geotriton  fuscus,  and  by  Payne  (1916)  for  certain  scorpions. 
The  mitochondrial  mass  forms  the  tail  sheath  in  such  spermatozoa.  It 
would  be  interesting  to  know  the  fate  of  the  plastids  in  spermatozoa 
formation  of  Ginkgo. 

The  similarity  of  behavior  of  the  chondriosomes  during  cell  division 
to  that  observed  for  the  starch-filled  plastids  of  Ginkgo  indicates  that 
the  distributing  mechanism  is  very  similar  in  each  case.  It  does  not 
seem  necessary  to  postulate  a  separate  mechanism  for  this  purpose,  the 
forces  already  in  action  being  of  a  type  which  would,  it  seems  to  me, 
bring  about  essentially  the  observed  distribution.  Whatever  the  force 
or  forces  may  be  by  which  the  chromosomes  are  distributed  during 
reduction,  the  direction  of  movement  of  the  chromosomes  shows  the 
direction  in  which  these  forces  act.  One  would  expect  that  a  large 
number  of  movable  cytoplasmic  structures  or  inclusions  would  be 
equally  distributed  between  the  cell  wall  and  the  nucleus,  if  in-  and 
out-going  currents  maintained  an  equilibrium  during  the  early  pro- 
phase. After  the  spindle  has  formed,  and  the  cell  is  a  bipolar  structure, 
the  forces  (which  we  may  think  of  as  currents)  move  toward  the  poles, 
and  presumably  back  toward  the  equator.  The  changes  of  position 
of  the  plastids  at  this  stage  indicate  such  lines  of  force.  As  the  nuclei 
grow,  apparently  by  taking  in  fluid,  their  increase  in  size  would  also 
tend  to  force  the  plastids  out  of  the  polar  and  into  the  equatorial 


246  University  of  California  Publications  in  Agricultural  Sciences       [Vol.  2 

regions.  The  barrel-shaped  spindle  would  hold  them  in  ring  formation. 
When  the  nuclei  have  attained  their  final  size,  the  plastids,  now  filled 
with  starch,  form  two  rows  with  the  cell  plate  between  them.  With  the 
formation  of  the  second  spindles  this  line  of  division  is  obliterated. 
It  is  possible  either  that  the  processes  involved  in  cell  plate  formation 
produce  the  line  of  division,  or  that  it  results  from  the  action  of  the 
opposing  forces  concerned  in  chromosome  division.  In  any  case,  the 
distortion  of  the  ring  at  late  second  ana-  and  early  telophase  might 
result  from  the  pull  of  the  same  forces  which  separated  the  chromo- 
somes. The  final  division  of  the  plastids  into  four  groups  may  depend 
somewhat  upon  the  invaginations  which  give  rise  to  the  lateral  Avails 
of  the  pollen  case.  In  the  pollen  cell  the  plastids  again  revert  to  the 
position  which  they  had  at  early  prophase  of  the  pollen  mother  cell. 


1924]  Mann:  Microsporogenesis  of  Ginkgo  biloba  L.  247 


LITERATURE  CITED 
Cardiff,  I.  D. 

1906.  A   study   of  synapsis  and   reduction.     Bull.   Torr.  Bot.  Club,  vol.  33, 

pp.  271-303. 

ISHIKAWA,  M. 

1910.     Uber  die  Zahl  der  Chromosomen  von  Ginkgo  biloba  L.    The  Bot.  Mag., 
Tokyo,  vol.  24,  pp.  225-226. 

JURANYI,  L. 

1872.     Ubei'  den  Bau  und  die  Entwickelung  des  Pollens  bei  Ceratozamia  longi- 
folia  Miq.     Jahrb.  f.  Wiss.  Bot.,  vol.  8,  pp.  382-400,  pis.  31-34. 
Moore,  A.  C. 

1903.     The  mitosis  in  the  spore  mother  cell  of  Pallavincinia.    Bot.  Gaz.,  vol. 
36,  pp.   384-388. 
Payne,  F. 

1916.     Germ  cells  of  Gryllotalpa.     Jour.  Morph.,  vol.  28,  pp.  287-327. 

Smith,  F.  G. 

1907.  Morphology   of  the   trunk   and   development   of  microsporangium    of 

Cycas.    Bot.  Gaz.,  vol.  43,  pp.  187-204,  pi.  10. 
Sprecher,  A. 

1907.     Le  Ginkgo  biloba  L.     (Geneva)  207  pp. 

Tern  i,  T. 

1914.     Condriosomi,  idiozoma  e  formazioni  periidiozomiche  nella   spermato- 
genesi   degli   Anfibrii.      (Ricerche   sul   Geotriton  fuscus.)      Arch.   f. 
Zellf.,  vol.  12,  pp.  1-96,  pis.  1-7. 
Wilson,  E.  B. 

1916.     The  distribution  of  the  chondriosomes  to  the  spermatozoa  of  scorpions. 
Science,  vol.  43,  p.  539. 


PLATE   44 

The  drawings  are  semi-diagrammatic.  They  were  made  with  a  camera  lucida, 
using  a  4  mm.  dry  objective  and  a  number  18  Zeiss  compensating  ocular.  The 
chromosomes  and  nuclei  are  simply  outlined,  the  plastids  at  the  upper  focus 
are  in  gray  wash  with  a  stippled  margin,  while  the  lower  ones  are  left  white. 
The  drawings  show  the  position  of  the  plastids  at  successive  stages  in  micro- 
sporogenesis. 

1.  Polar  view  of  late  first  prophase  showing  12  pairs  of  chromosomes,  one  of 
which  is  about  twice  the  size  of  the  others. 

2.  Lateral  view  of  same  stage  showing  that  the  spindle  area  is  largely  free 
of  plastids.     Most  of  the  plastids  are  near  the  poles. 

3.  Lateral  view  of  late  anaphase  showing  movement  of  plastids  from  poles 
toward  equator  of  cell. 

1.  Late  telophase.  The  plastids  are  now  arranged  in  a  double  ring  at  the 
equator. 

5.  Late  second  prophase.  The  plastids  are  so  closely  massed  that  ring 
formation  cannot   be  distinguished. 

6.  Late  second  anaphase.     The  ring  of  plastids  is  being  distorted. 

7.  Second  telophase.  The  ring  is  now  breaking  into  four  segments,  one  group 
lying  about  the  inner  face  of  each  of  the  four  nuclei. 

8.  Early  cell  division.  The  dotted  lines  show  cell  plate.  The  outer  wall  is 
pushing  inward  between  each  of  the  four  nuclei. 

9.  Cell  division  completed. 


[248] 


UNIV.    CALIF.    PUBL.    AGRI.    SCI.    VOL.    2 


[  MANN  |    PLATE    44 


